Free vitamin D is independently associated with systolic blood pressure in diabetic patients with impaired kidney function.

Vitamin D contributes to blood pressure (BP) regulation. We compared the association of BP in diabetic patients with either total vitamin D - the standard way of analyzing the vitamin D status - or free vitamin D, because only free vitamin D passes the cell membrane and interacts with the nuclear vitamin D receptor (VDR). An analytical cross-sectional study was conducted with 178 diabetic patients with impaired kidney function. Free and total vitamin D concentrations were measured in all patients. Multiple linear regression analysis considering patient age, sex, body mass index, height, smoking and drinking situation, the use of antihypertensive drugs, cholecalciferol treatment, C-reactive protein and estimated glomerular filtration rate as confounding factors were conducted to compare the association of free and total vitamin D with systolic and diastolic blood pressure (SBP and DBP). Multiple linear regression analysis revealed that neither SBP nor DBP was correlated with total vitamin D (SBP, 95% CI -0.405 ~ 0.159, p = 0.390; DBP, 95% CI -0.131 ~ 0.142, p = 0.933) (Table 2). However, the concentration of free vitamin D was independently associated with SBP (95% CI -2.691 ~ -0.210; p = 0.022) (Table 3), but not with DBP (95% CI -0.934 ~ 0.285; p = 0.293). In conclusion, free - but not total - vitamin D serum concentrations in patients with diabetes and impaired kidney function are inversely correlated with SBP. This study suggests that free vitamin D measurements might be more clinically relevant - as compared to measurements of total vitamin D - to adjust vitamin D therapy in diabetic patients with impaired kidney function.

Osteoprotegerin is an independent risk factor predicting death in stable renal transplant recipients.

BACKGROUND: Vascular calcification is common in chronic kidney disease and is associated with significant cardiovascular morbidity and mortality. One of the important factors regulating vascular calcification is osteoprotegerin (OPG). There are, however, limited data on the impact of OPG on all-cause mortality and graft loss in kidney transplant recipients so far. Given its impact on vascular calcification, the aim of our study is to analyze whether OPG was a risk factor of all-cause mortality and graft loss in 600 stable kidney transplant recipients. MATERIALS AND METHODS: 600 stable renal transplant recipients (367 women, 233 men) were followed for all-cause mortality and graft loss for 3 years. Blood and urine samples for analysis and clinical data were collected at study entry. We performed Kaplan-Meier survival analysis and Cox regression models considering confounding factors such as age, estimated glomerular filtration rate (eGFR), cold ischemia time, HbA1c, phosphorus, calcium, and albumin. RESULTS: 65 patients died, and 38 patients had graft loss during the observation period. The OPG baseline concentrations had no effect on graft loss, whereas Kaplan-Meier survival curve showed that baseline plasma OPG concentrations were associated with all-cause mortality in stable kidney transplant recipients (p < 0.0001, log-rank test). After multiple Cox regression analysis adjusting for age, eGFR, cold ischemia time, HbA1c, phosphorus, calcium, and albumin, plasma levels of OPG remained an independent predictor of all-cause mortality (HR, 1.181; 95%CI 1.035 - 1.347; p = 0.014). CONCLUSION: Baseline plasma OPG is an independent risk factor for all-cause mortality but not graft loss in patients after kidney transplantation.

Urinary Neutrophil Gelatinase-Associated Lipocalin/Hepcidin-25 Ratio for Early Identification of Patients at Risk for Renal Replacement Therapy After Cardiac Surgery: A Substudy of the BICARBONATE Trial.

BACKGROUND: Acute kidney injury requiring renal replacement therapy (AKI-RRT) is strongly associated with mortality after cardiac surgery; however, options for early identification of patients at high risk for AKI-RRT are extremely limited. Early after cardiac surgery, the predictive ability for AKI-RRT even of one of the most extensively evaluated novel urinary biomarkers, neutrophil gelatinase-associated lipocalin (NGAL), appears to be only moderate. We aimed to determine whether the NGAL/hepcidin-25 ratio (urinary concentrations of NGAL divided by that of hepcidin-25) early after surgery may compare favorably to NGAL for identification of high-risk patients after cardiac surgery. METHODS: This is a prospective substudy of the BICARBONATE trial, a multicenter parallel-randomized controlled trial comparing perioperative bicarbonate infusion for AKI prevention to usual patient care. At a tertiary referral center, 198 patients at increased kidney risk undergoing cardiac surgery with cardiopulmonary bypass were included into the present study. The primary outcome measure was defined as AKI-RRT. Secondary outcomes were in-hospital mortality and long-term mortality. We compared area under the curve of the receiver operating characteristic (AUC-ROC) of urinary NGAL with that of the urinary NGAL/hepcidin-25 ratio within 60 minutes after end of surgery. We compared adjusted AUC and performed cross-validated reclassification statistics of the (logarithmic) urinary NGAL/hepcidin-25 ratio adjusted to Cleveland risk score/EuroScore, cross-clamp time, age, volume of packed red blood cells, and (logarithmic) urinary NGAL concentration. The association of the NGAL/hepcidin-25 ratio with long-term patient survival was assessed using Cox proportional hazard regression analysis adjusting for EuroScore, aortic cross-clamp time, packed red blood cells and urinary NGAL. RESULTS: Patients with AKI-RRT (n = 13) had 13.7-times higher NGAL and 3.3-times lower hepcidin-25 concentrations resulting in 46.9-times higher NGAL/hepcidin-25 ratio early after surgery compared to patients without AKI-RRT. The NGAL/hepcidin-25 ratio had higher AUC-ROC compared with NGAL for risk of AKI-RRT and in-hospital mortality (unadjusted AUC-ROC difference 0.087, 95% confidence interval [CI], 0.036-0.138, P < .001; 0.082, 95% CI, 0.018-0.146, P = .012). For AKI-RRT, the NGAL/hepcidin-25 ratio increased adjusted category-free net reclassification improvement (cfNRI; 0.952, 95% CI, 0.437-1.468; P < .001) and integrated discrimination improvement (IDI; 0.040, 95% CI, 0.008-0.073; P = .016) but not AUC difference. For in-hospital mortality, the ratio improved AUC of the reference model (AUC difference 0.056, 95% CI, 0.003-0.108; P = .037) and cfNRI but not IDI. The urinary NGAL/hepcidin-25 ratio remained significantly associated with long-term mortality after adjusting for the model covariates. CONCLUSIONS: The urinary NGAL/hepcidin-25 ratio appears to early identify high-risk patients and outperform NGAL after cardiac surgery. Confirmation of our findings in other cardiac surgery centers is now needed.

C-terminal and intact FGF23 in kidney transplant recipients and their associations with overall graft survival.

BACKGROUND: Increased fibroblast growth factor 23 (FGF23) is a risk factor for mortality, cardiovascular disease, and progression of chronic kidney disease. Limited data exist comparing the association of either c-terminal FGF23 (cFGF23) or intact FGF23 (iFGF23) in kidney transplant recipients (KTRs) with overall (all-cause) graft loss. METHODS: We conducted a prospective observational cohort study in 562 stable kidney transplant recipients. Patients were followed for graft loss and all-cause mortality for a median follow-up of 48 months. RESULTS: During a median follow-up of 48 months, 94 patients had overall graft loss (primary graft loss or death with functioning graft). Both cFGF23 and iFGF23 concentrations were significantly higher in patients with overall graft loss than those without (24.59 [11.43-87.82] versus 10.67 [5.99-22.73] pg/ml; p < 0.0001 and 45.24 [18.63-159.00] versus 29.04 [15.23-60.65] pg/ml; p = 0.002 for cFGF23 and iFGF23, respectively). Time-dependent ROC analysis showed that cFGF23 concentrations had a better discriminatory ability than iFGF23 concentrations in predicting overall (all-cause) graft loss. Cox regression analyses adjusted for risk factors showed that cFGF23 (HR for one unit increase of log transformed cFGF23: 1.35; 95% CI, 1.01-1.79; p = 0.043) but not iFGF23 (HR for one unit increase of log transformed iFGF23: 0.97; 95% CI, 0.75-1.25; p = 0.794) was associated with the overall graft loss. CONCLUSION: Elevated cFGF23 concentrations at baseline are independently associated with an increased risk of overall graft loss. iFGF23 measurements were not independently associated with overall graft loss. The cFGF23 ELISA might detect bioactive FGF23 fragments that are not detected by the iFGF23 ELISA.

Inferior vena cava-syndrome.

Inferior vena cava syndrome (IVCS) is caused by agenesis, compression, invasion, or thrombosis of the IVC, or may be associated with Budd-Chiari syndrome. Its incidence and prevalence are unknown. Benign IVCS is separated from malignant IVCS. Both cover a wide clinical spectrum reaching from asymptomatic to highly symptomatic cases correlated to the underlying cause, the acuity, the extent of the venous obstruction, and the recruitment and development of venous collateral circuits. Imaging is necessary to determine the underlying cause of IVCS and to guide clinical decisions. Interventional therapy has changed the therapeutic approach in symptomatic patients. This article provides an overview over IVCS and focuses on interventional therapeutic methods and results.

Endostatin Is an Independent Risk Factor of Graft Loss after Kidney Transplant.

BACKGROUND: Endostatin is a 20-kDa C-terminal fragment of collagen XVIII, known for its ability to inhibit the proliferation of capillary endothelial cells. Previous studies suggested that circulating endostatin independently predicts incident chronic kidney disease. However, the impact of endostatin on graft loss level in kidney transplant recipients (KTRs) remains unknown. METHODS: We conducted a prospective observational cohort study in 574 maintenance KTRs. Patients were followed for kidney graft loss and all-cause mortality during a median follow-up of 48 months. Serum-, and urine-samples and clinical data were collected at baseline. Serum Endostatin concentration was analyzed by an ELISA. RESULTS: Among 574 patients, 37 patients had graft loss and 62 patients died. For graft loss, the optimal cut-off value based on receiver operating characteristics analysis (area under the curve 0.79, 95% CI 0.71-0.86, p < 0.001) of endostatin was 147.3 pmol/L. Kaplan-Meier curves revealed that higher serum endostatin concentrations positively correlated with graft loss (p < 0.001). Multivariable Cox regression analyses showed that baseline endostatin concentrations were significantly associated with graft loss after adjusting for graft loss risk factors (adjusted hazard ratio [HR] 8.34; 95% CI 2.19-31.72; p = 0.002). The adjusted HRs for classical graft loss risk factors such as baseline estimated glomerular filtration rate and urinary protein excretion were lower (1.91 and 5.44, respectively). In contrast to graft loss, baseline endostatin concentrations were not associated with all-cause mortality. CONCLUSION: Increased serum endostatin at baseline is independently associated with the risk of graft loss in KTRs.

Superior vena cava syndrome.

The superior vena cava syndrome (SVCS) is caused by compression, invasion, and/or thrombosis of the superior vena cava and/or the brachiocephalic veins. Benign SVCS is separated from malignant SVCS. SVCS comprises a broad clinical spectrum reaching from asymptomatic cases to rare life-threatening emergencies with upper airway obstruction and increased intracranial pressure. Symptoms are correlated to the acuity and extent of the venous obstruction and inversely correlated to the development of the venous collateral circuits. Imaging is necessary to determine the exact underlying cause and to guide further interventions. Interventional therapy has widely changed the therapeutic approach in symptomatic patients. This article provides an overview over this complex syndrome and focuses on interventional therapeutic methods and results.

Free 25-Vitamin D Is Correlated with Cardiovascular Events in Prevalent Hemodialysis Patients but Not with Markers of Renal Mineral Bone Disease.

Free vitamin D is the biologically active form of vitamin D. Vitamin D deficiency is associated with cardiovascular disease, the most common cause of mortality in hemodialysis patients. The goal of our current study was to investigate the relation between blood concentrations of free 25-hydroxyvitamin D with cardiovascular events in end-stage chronic kidney disease patients on hemodialysis, because this is unknown so far. We measured free vitamin D levels in 117 stable consecutive prevalent patients in September as a surrogate of vitamin D exposure during the past 6 months, and recorded the number of cardiovascular events during the previous 6 months defined as hospitalization due to heart failure, episodes of acute coronary syndrome, and stroke. Fourteen events occurred during the observation period. In patients without any cardiovascular events the free vitamin D levels were significantly higher as compared to those with cardiovascular events (patients without events: 5.68 [4.37-9.27] pg/mL; patients with events: 4.74 [3.46-5.37] pg/mL, p = 0.015). This finding remained stable after multiple regression analysis considering confounding factors such as age, time on dialysis, preexisting diabetes, hypertension, and coronary heart disease. In conclusion, our study shows that free vitamin D serum concentrations are independently associated with major cardiovascular events in chronic kidney disease patients on dialysis.

[Treatment, clinical course, and cross-sectoral information transmission in patients with acute-on-chronic kidney injury]. / Behandlung, klinischer Verlauf und sektorenübergreifende Informationsübermittlung bei Patienten mit akut-auf-chronischer Nierenschädigung.

BACKGROUND: Delayed diagnosis and undertherapy of acute-on-chronic kidney injury (AKI-on-CKD) may trigger multiple organ injury and worsen clinical outcome. OBJECTIVES: This study focused on description of in-hospital care and cross-sectoral information transmission of patients with AKI-on-CKD including subgroup analyses (under surgical vs. non-surgical and nephrology vs. non-nephrology care). MATERIALS AND METHODS: At a university clinic, we analysed clinical measures and documentation in patients with AKI-on-CKD. Cox regression was performed to identify independent risk factors for in-hospital-mortality and 180-day mortality. RESULTS: In 38 (25.3%) of 150 patients, progressing AKI-on-CKD was found. Nineteen patients (12.7%) received acute dialysis. Thirty patients (20.0%) died in hospital. Systemic hypotension (n = 76, 50.7%) and nephrotoxins (n = 26, 17.3%), both considered as causes for AKI-on-CKD, were treated in 36.8 and 19.2%, respectively, of affected patients. Fluid balance was documented in one third of patients. Nephrology referral was requested in 38 (25.3%) of patients (median 24.0 h after AKI-on-CKD start). Acute renal complications (n = 74, 49.3%) were an independent risk factor for in-hospital mortality (ExpB 6.5, p = 0.022) or 180-day mortality (ExpB 3.3, p = 0.034). Rarely, outpatient physicians were informed about AKI-on-CKD (n = 42, 28.0%) or renal function follow-up was recommended (n = 14, 11.7% of surviving patients). CONCLUSIONS: Care gaps in therapy and cross-sectoral information transmission in patients with AKI-on-CKD were identified.

A patient with a floating kidney and nephrotic syndrome.

Asymptomatic retroperitoneal fluid is rarely detected. We encountered a young man with known nephrotic syndrome who presented with left-sided abdominal pain. He had very little peripheral edema, but a massive fluid collection around a "floating" left kidney, as determined by three different imaging studies. Large amounts of fluid and a cyst surrounding the kidney were removed laparascopically and malignancy was excluded. The patient had a rare benign cystic mesothelioma together with focal-segmental glomerulosclerosis. Both diseases do not seem to be causally related. Complete surgical resection of the mesothelioma is the definitive treatment. If resection is incomplete, recurrence is frequent. Indeed, when our patient returned some years later for an elective hernia repair, the fluid collection had recurred.

G-protein coupled receptor auto-antibodies in thromboangiitis obliterans (Buerger's disease) and their removal by immunoadsorption.

BACKGROUND: Immunhistopathological and serological data favors an immunopathogenesis of thromboangiitis onliterans (TAO, Buerger's disease). Auto antbodies seem to play a major role. Immunoadsorption (IA) proved to be therapeutically effective. We focused on agonistic autoantibodies (agAAB) directed against G-protein coupled receptors (GPCR) and proved the hypothesis, that these agAAB might be present in TAO and that a five day course of IA might be able to eliminate these agAAB effectively. PATIENTS AND METHODS: Between December 2012 and May 2014 11 TAO-patients were treated by IA in a five day course. AgAAB-analysis was performed using specific ELISA techniques. RESULTS: AgAAB were detected in 9 out of 11 patients (81.8 %).Multiple agAAB were present in 7 patients (63.6 %). A clustering of agAAB directed against loop1 of the adrenergic α1-receptor and the endothelin-A-(ETA)receptor was identified, representing 72.7 % resp. 54.5 % of the patients. AgAAB directed against the angiotensin-1 (AT-1) epitope 1 or 2 were detected in 3 patients and agAAB directed against protease-activated receptor (PAR) loop1/2 were seen in 2 patients. AgAAB directed against ETA-receptor loop1 never appeared without agAAB directed against α1-receptor loop1. Immediately after a five day-course of IA agAAB were absent in 81.8 % of the total study group and in 77.8 % of all cases tested positive for agAAB before IA. CONCLUSIONS: AgAAB directed against GPCR were identified in TAO patients with a clustering of agAAB directed against α-1-adrenergic receptor loop1 and ETA-receptor loop1. AgAA were eliminated by IA in the majority of cases. We suggest that these agAA play an important role in the pathogenesis of TAO and that their elimination might be responsible for the positive therapeutic effects reported in patients treated with IA.

Immune response of heterologous versus homologous prime-boost regimens with adenoviral vectored and mRNA COVID-19 vaccines in immunocompromised patients.

Due to rare but major adverse reactions to the AstraZeneca adenoviral ChAdOx1-S-nCoV-19 vaccine (ChAd), German health authorities recommended adults under 60 who received one dose of ChAd, to receive a second dose of the BioNTech mRNA BNT162b2 vaccine (BNT) as a booster. Studies in the general population suggest an enhanced efficacy of the heterologous (ChAd-BNT) compared to the homologous (BNT-BNT) vaccination regimen. However, an analysis of the efficacy in patient populations with a high risk of severe COVID-19 due to acquired immunodeficiency is still missing. We therefore compared both vaccination regimens in healthy controls, patients with gynecological tumors after chemotherapy, patients on dialysis and patients with rheumatic diseases concerning the humoral and cellular immune response. The humoral and cellular immune response differed substantially in healthy controls compared to patients with acquired immunodeficiency. Overall, the most significant differences between the two immunization regimens were found in neutralizing antibodies. These were always higher after a heterologous immunization. Healthy controls responded well to both vaccination regimens. However, the formation of neutralizing antibodies was more pronounced after a heterologous immunization. Dialysis patients, on the other hand, only developed an adequate humoral and particularly cellular immune response after a heterologous immunization. Tumor and rheumatic patients also - to a weaker extent compared to dialysis patients - benefited from a heterologous immunization. In conclusion, the heterologous COVID-19 vaccination regimens (ChAd-BNT) seem to have an advantage over the homologous vaccination regimens, especially in immunocompromised patients such as patients with end-stage kidney disease treated with hemodialysis.

Urinary T Cells Identify Renal Antineutrophil Cytoplasmic Antibody-Associated Vasculitis and Predict Prognosis: A Proof of Concept Study.

Introduction: Necrotizing crescentic glomerulonephritis is a major contributor to morbidity and mortality in Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). Because therapy relies on immunosuppressive agents with potentially severe adverse effects, a reliable noninvasive biomarker of disease activity is needed to guide treatment. Methods: We used flow cytometry to quantify T cell subsets in blood and urine samples from 95 patients with AAV and 8 controls to evaluate their biomarker characteristics. These were compared to soluble markers, monocyte chemoattractant protein-1 (MCP-1), soluble CD163 (sCD163), soluble CD25 (sCD25), and complement C5a (C5a), measured using multiplex analysis. Available kidney biopsies (n = 21) were classified according to Berden. Results: Patients with active renal AAV (rAAV) showed significantly higher urinary cell counts than those in remission, or those with extrarenal manifestation, or healthy controls. Urinary T cells showed robust discrimination of disease activity with superior performance compared to MCP-1 and sCD163. Patients whose kidney biopsies had been classified as "crescentic" according to Berden classification showed higher urinary T cell counts. Discordant regulatory T cells (Treg) proportions and CD4+/CD8+ ratio in blood and urine suggested that urinary cells reflect tissue migration rather than mere micro-bleeding. Furthermore, urinary Treg and T helper cells (TH17) patterns were associated with clinical response and risk of renal relapse. Conclusion: Urinary T cells reflect the renal inflammatory milieu in AAV and provide further insights into the pathogenesis of this chronic condition. Their promising potential as noninvasive diagnostic and prognostic biomarkers deserves further exploitation.

NGAL/hepcidin-25 ratio and AKI subtypes in patients following cardiac surgery: a prospective observational study.

BACKGROUND: Acute kidney injury (AKI) subtypes combining kidney functional parameters and injury biomarkers may have prognostic value. We aimed to determine whether neutrophil gelatinase-associated lipocalin (NGAL)/hepcidin-25 ratio (urinary concentrations of NGAL divided by that of hepcidin-25) defined subtypes are of prognostic relevance in cardiac surgery patients. METHODS: We studied 198 higher-risk cardiac surgery patients. We allocated patients to four groups: Kidney Disease Improving Global Outcomes (KDIGO)-AKI-negative and NGAL/hepcidin-25 ratio-negative (no AKI), KDIGO AKI-negative and NGAL/hepcidin-25 ratio-positive (subclinical AKI), KDIGO AKI-positive and NGAL/hepcidin-25 ratio-negative (clinical AKI), KDIGO AKI-positive and NGAL/hepcidin-25 ratio-positive (combined AKI). Outcomes included in-hospital mortality (primary) and long-term mortality (secondary). RESULTS: We identified 127 (61.6%) patients with no AKI, 13 (6.6%) with subclinical, 40 (20.2%) with clinical and 18 (9.1%) with combined AKI. Subclinical AKI patients had a 23-fold greater in-hospital mortality than no AKI patients. For combined AKI vs. no AKI or clinical AKI, findings were stronger (odds ratios (ORs): 126 and 39, respectively). After adjusting for EuroScore, volume of intraoperative packed red blood cells, and aortic cross-clamp time, subclinical and combined AKI remained associated with greater in-hospital mortality than no AKI and clinical AKI (adjusted ORs: 28.118, 95% CI 1.465-539.703; 3.737, 95% CI 1.746-7.998). Cox proportional hazard models found a significant association of biomarker-informed AKI subtypes with long-term survival compared with no AKI (adjusted ORs: pooled subclinical and clinical AKI: 1.885, 95% CI 1.003-3.542; combined AKI: 1.792, 95% CI 1.367-2.350). CONCLUSIONS: In the presence or absence of KDIGO clinical criteria for AKI, the urinary NGAL/hepcidin-25-ratio appears to detect prognostically relevant AKI subtypes. TRIAL REGISTRATION NUMBER: NCT00672334, clinicaltrials.gov, date of registration: 6th May 2008, https://clinicaltrials.gov/ct2/show/NCT00672334 . Definition of AKI subtypes: subclinical AKI (KDIGO negative AND Ratio-positive), clinical AKI (KDIGO positive AND Ratio-negative) and combined AKI (KDIGO positive AND Ratio-positive) with urinary NGAL/hepcidin-25 ratio-positive cut-off at 85% specificity for in-hospital death. AKI, acute kidney injury. AUC, area under the curve. NGAL, neutrophil gelatinase-associated lipocalin. KDIGO, Kidney Disease Improving Global Outcomes Initiative AKI definition.

Impact of hypertension on long-term humoral and cellular response to SARS-CoV-2 infection.

It was shown that hypertension delays SARS CoV-2 viral clearance and exacerbates airway hyperinflammation in the respiratory tract. However, it is unknown whether hypertension determines the long-term cellular and humoral response to SARS Cov2. Health care workers (HCWs) after an outbreak of SARS Cov-2 infections were analyzed. Infected HCWs were not vaccinated before blood collection. 5-14 months (median 7 months) after detection of SARS CoV-2 infection, blood was taken to analyze humoral response (S1 IgG and SARS CoV-2 neutralizing antibodies) and cellular (T cell responses to SARS-CoV-2 with Lymphocyte Transformation Test). To identify clinical factors that determine the immune response, a multivariate regression analysis was done considering age, BMI, sex, diabetes, hypertension, smoking, COPD, asthma and time between PCR positivity and blood collection as confounding factors. Infected hypertensive HCWs more often needed to be hospitalized than non-hypertensive HCWs, but were less likely to develop anosmia and myalgia. The long-term humoral and cellular immune response was significantly strengthened in hypertensive versus normotensive infected HCWs. Multivariate regression analysis revealed that hypertension was independently associated with the humoral response to SARS CoV-2 infection. Multivariate regression analysis using same confounding factors for the humoral response showed a clear trend for an association with the cellular response to SARS CoV-2 infection as well. In conclusion, SARS CoV-2 infection strengthened immune response to SARS CoV-2 infection in hypertensive HCWs independent of other risk factors.

T-cell proliferation assay for the detection of SARS-CoV-2-specific T-cells.

Both infection with and vaccination against SARS-CoV-2 trigger a complex B-cell and T-cell response. Methods for the analysis of the B-cell response are now well established. However, reliable methods for measuring the T-cell response are less well established and their usefulness in clinical settings still needs to be proven. Here, we have developed and validated a T-cell proliferation assay based on 3H thymidine incorporation. The assay is using SARS-CoV-2 derived peptide pools that cover the spike (S), the nucleocapsid (N) and the membrane (M) protein for stimulation. We have compared this novel SARS-CoV-2 lymphocyte transformation test (SARS-CoV-2 LTT) to an established ELISA assay detecting Immunoglobulin G (IgG) antibodies to the S1 subunit of the SARS-CoV-2 spike protein. The study was carried out using blood samples from both vaccinated and infected health care workers as well as from a non-infected control group. Our novel SARS-CoV-2 LTT shows excellent discrimination of infected and/or vaccinated individuals versus unexposed controls, with the ROC analysis showing an area under the curve (AUC) of > 0.95. No false positives were recorded as all unexposed controls had a negative LTT result. When using peptide pools not only representing the S protein (found in all currently approved vaccines) but also the N and M proteins (not contained in the vast majority of vaccines), the novel SARS-CoV-2 LTT can also discriminate T-cell responses resulting from vaccination against those induced by infection.

Head-to-head comparison of two SGLT-2 inhibitors on AKI outcomes in a rat ischemia-reperfusion model.

The CREDENCE trial testing canagliflozin and the EMPA-REG OUTCOME trial testing empagliflozin suggest different effects on acute kidney injury (AKI). AKI diagnosis was mainly made based on changes of serum creatinine (sCr) although this also reflect mode of action of SGLT-2 inhibitors. We analyzed both compounds in a rat AKI model. The renal ischemia-reperfusion injury (I/R) model was used. Four groups were analyzed: sham, I/R+placebo, I/R+canagliflozin (30 mg/kg/day), I/R+ empagliflozin (10 mg/kg/day). Glucose excretion was comparable in both treatment groups indicating comparable SGLT-2 inhibition. Comparing GFR surrogate markers after I/R (sCr and blood urea nitrogen (BUN)), sCr peaked 24 h after I/R, BUN after 48 h, respectively, in the placebo treated I/R group. At all investigated time points after I/R sCr and BUN was higher in the I/R + canagliflozin group as compared to placebo treated rats, whereas the empagliflozin group did not differ from the placebo group. I/R led to tubular dilatation and necrosis. Empagliflozin was able to reduce that finding whereas canagliflozin had no effect. Treatment with empagliflozin also resulted in a significant reduction in an improved inflammatory score (p = 0.006). Renal expression of kidney injury molecule-1 (KIM-1) increased after I/R and empagliflozin but not canagliflozin significantly alleviated KIM-1 expression. I/R reduced urinary miR-26a excretion. Empagliflozin but not canagliflozin was able to restore normal levels of urinary miR-26a. This study in an AKI model confirmed safety data in the EMPA-REG OUTCOME trial suggesting that empagliflozin might reduce AKI risk. The empagliflozin effects on KIM-1 and miR-26a might indicate beneficial regulation of inflammation. These data should stimulate clinical studies with AKI risk as primary endpoint.

Protective α1-antitrypsin effects in autoimmune vasculitis are compromised by methionine oxidation.

BackgroundAntineutrophil cytoplasmic autoantibody-associated (ANCA-associated) vasculitidies (AAV) are life-threatening systemic autoimmune conditions. ANCAs directed against proteinase 3 (PR3) or myeloperoxidase (MPO) bind their cell surface-presented antigen, activate neutrophils, and cause vasculitis. An imbalance between PR3 and its major inhibitor α1-antitrypsin (AAT) was proposed to underlie PR3- but not MPO-AAV. We measured AAT and PR3 in healthy individuals and patients with AAV and studied protective AAT effects pertaining to PR3- and MPO-ANCA.MethodsPlasma and blood neutrophils were assessed for PR3 and AAT. WT, mutant, and oxidation-resistant AAT species were produced to characterize AAT-PR3 interactions by flow cytometry, immunoblotting, fluorescence resonance energy transfer assays, and surface plasmon resonance measurements. Neutrophil activation was measured using the ferricytochrome C assay and AAT methionine-oxidation by Parallel Reaction Monitoring.ResultsWe found significantly increased PR3 and AAT pools in patients with both PR3- and MPO-AAV; however, only in PR3-AAV did the PR3 pool correlate with the ANCA titer, inflammatory response, and disease severity. Mechanistically, AAT prevented PR3 from binding to CD177, thereby reducing neutrophil surface antigen for ligation by PR3-ANCA. Active patients with PR3-AAV showed critical methionine-oxidation in plasma AAT that was recapitulated by ANCA-activated neutrophils. The protective PR3-related AAT effects were compromised by methionine-oxidation in the AAT reactive center loop but preserved when 2 critical methionines were substituted with valine and leucine.ConclusionPathogenic differences between PR3- and MPO-AAV are related to AAT regulation of membrane-PR3, attenuating neutrophil activation by PR3-ANCA rather than MPO-ANCA. Oxidation-resistant AAT could serve as adjunctive therapy in PR3-AAV.FUNDINGThis work was supported by KE 576/10-1 from the Deutsche Forschungsgemeinschaft, SCHR 771/8-1 from the Deutsche Forschungsgemeinschaft, grant 394046635 - SFB 1365 from the Deutsche Forschungsgemeinschaft, and ECRC grants.